Optimization of antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof

ABSTRACT

The present invention provides isolated monoclonal antibodies that specifically bind LAG-3, and have optimized functional properties compared to previously described anti-LAG-3 antibodies, such as antibody 25F7 (US 2011/0150892 A1). These properties include reduced deamidation sites, while still retaining high affinity binding to human LAG-3, and physical (i.e., thermal and chemical) stability. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided, as well as immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies. The present invention also provides methods for detecting LAG-3, as well as methods for treating stimulating immune responses using an anti-LAG-3 antibody of the invention. Combination therapy, in which the antibodies are co-administered with at least one additional immunostimulatory antibody, is also provided.

RELATED APPLICATIONS

This application claims priority to PCT/US2013/048999, filed Jul. 2,2013, which claims priority to U.S. provisional application 61/667,058,filed Jul. 2, 2012. The contents of the foregoing applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 30, 2013, isnamed MXI-513_Sequence_Listing.txt and 41.986 KB in size.

BACKGROUND OF THE INVENTION

Therapeutic antibodies are one of the fastest growing segments of thepharmaceutical industry. To maintain potency (i.e., activity) andminimize immunogenicity, antibodies and other protein drugs must beprotected from physical and chemical degradation during manufacturingand storage. Indeed, one of the primary difficulties in developingantibody therapeutics is the potential immunogenic response whenadministered to a subject, which can lead to rapid clearance or eveninduce life-threatening side effects including anaphylactic shock.Various factors influence the immunogenicity of an antibody such as itsphysiochemical properties (e.g., purity, stability, or solubility),clinical factors (e.g., dose, route of administration, heterogeneity ofthe disease, or patient features), and concomitant treatment with otheragents (Swann et al. (2008) Curr Opinion Immuol 20:493-499).

Immunogenicity of antibodies and/or loss of antibody activity is oftendue to deamidation. Deamidation is a chemical degradative process thatspontaneously occurs in proteins (e.g., antibodies). Deamidation removesan amide functional group from an amino acid residue, such as asparagineand glutamine, thus damaging its amide-containing side chains. This, inturn, causes structural and biological alterations throughout theprotein, thus creating heterogeneous forms of the antibody. Deamidationis one of the most common post-translational modifications that occursin recombinantly produced therapeutic antibodies.

For example, heterogeneity in the heavy chain of monoclonal antibodyh1B4 (a humanized anti-CD18 antibody) due to deamidation during cellculture was reported by Tsai et al. (Pharm Res 10(11):1580 (1993)). Inaddition, reduction/loss of biological activity due to deamidation hasbeen a recognized problem. For example, Kroon et al. characterizedseveral deamidation sites in therapeutic antibody OKT3, and reportedthat samples of OKT3 production lots (aged 14 months to 3 years) hadfallen below 75% activity (Pharm Res 9(11):1386 (1992), page 1389,second column). In addition, samples of OKT3 showing large amounts ofthe oxidized peptides in their maps had significantly reduced activityin the antigen binding potency assay (page 1390, first column). Theauthors concluded that specific sites of chemical modification thatoccur upon storage of OKT3 were identified by peptide mapping andcorrelated with observed changes in chemical analyses and biologicalassays of the antibody (page 1392, first column). Loss of biologicalactivity also has been reported for a variety of other deamidatedtherapeutic proteins, including recombinant human DNase (Cacia et al.(1993) J. Chromatogr. 634:229-239) and recombinant soluble CD4 (Teshimaet al. (1991) Biochemistry 30:3916-3922).

Overall, deamidation poses a significant and unpredictable problem tothe pharmaceutical industry. Efforts associated with monitoring thevariability caused by deamidation within antibody therapeutics, inparticular, as well as FDA concerns associated with this variability,increase costs and delay clinical trials. Moreover, modifications toaddress this issue, including shifting conditions (e.g., temperature,pH, and cell type) associated with recombinant production and/oralteration of amino acids which are susceptible to deamidation (e.g.,site-directed mutagenesis) can negatively impact stability and activity,especially when changes are made within the complementarity determiningregions (CDRs) of the antibody. Accordingly, the need exists for morestable versions of therapeutic antibodies.

SUMMARY

The present invention provides isolated monoclonal antibodies (e.g.,human monoclonal antibodies) that bind LAG-3 (e.g., human LAG-3) andhave optimized physical stability compared to previously describedanti-LAG-3 antibodies. In particular, the invention relates to amodified form of antibody 25F7 (US 2011/0150892 A1) which exhibitssignificantly improved thermal and chemical stability compared to theunmodified antibody. Specifically, by altering the critical bindingregion of the heavy chain CDR2 domain of antibody 25F7, it was shownthat the modified antibody exhibited significantly higher physical andthermal stability, reduced deamidation, higher thermal reversibility,and lower aggregation. At the same time, it was unexpectedly observedthat the modified antibody retained the same high binding affinity tohuman LAG-3 and functional activity of the unmodified antibody,including the ability to inhibit binding of LAG-3 to majorhistocompatibility (MHC) Class II molecules and stimulateantigen-specific T cell responses. The combined substantial increase instability and retention of binding/biological activity of the modifiedantibody was surprising, particularly in view of the criticality of CDRsregions to antibody function.

The antibodies of the invention can be used for a variety ofapplications, including detection of LAG-3 protein and stimulation ofantigen-specific T cell responses in tumor-bearing or virus-bearingsubjects.

Accordingly, in one aspect, the invention pertains to an isolatedmonoclonal antibody (e.g., a human antibody), or an antigen-bindingportion thereof, having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 12. In another embodiment, theantibody further includes a light chain variable region comprising theamino acid sequence of SEQ ID NO: 14. In another embodiment, theantibody, or antigen-binding portion thereof, includes the CDR1, CDR2,and CDR3 regions of a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 12 (e.g., SEQ ID NOs: 15, 16, and 17,respectively). In another embodiment, the antibody further includes theCDR1, CDR2, and CDR3 regions of a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 12 (e.g., SEQ ID NOs: 18, 19, and20, respectively).

In a preferred embodiment the antibody exhibits increased physicalproperties (i.e., thermal and chemical stability) compared to antibody25F7, while still retaining at least the same binding affinity for humanLAG-3 as 25F7. For example, the antibody exhibits decreased sequencevariability in the heavy chain CDR2 region due to deamidation, comparedto antibody 25F7, e.g., approximately 2.5% or less modification of theamino acid sequence after 12 weeks at 4° C. (i.e., under “real-time”stability studies as described herein) and/or approximately 12.0% orless modification of the amino acid sequence after 12 weeks at 40° C.(i.e., under accelerated stress conditions, as described herein), whilestill retaining a binding affinity for human LAG-3 of about at leastK_(D) of 1×10⁻⁷ M or less (more preferably, a K_(D) of 1×10⁻⁸ M or less,a K_(D) of 5×10⁻⁹ M or less, or a K_(D) of 1×10⁻⁹ M or less). In anotherembodiment, the antibody exhibits thermal reversibility of at leastabout 40% in PBS at pH8.0.

In another embodiment, the antibody possesses a higher meltingtemperature (indicating greater overall stability in vivo), compared tothe unmodified antibody (Krishnamurthy R and Manning M C (2002) CurrPharm Biotechnol 3:361-71). In one embodiment, the antibody exhibits aT_(M1) (the temperature of initial unfolding) of greater than 60° C.,e.g., greater than 65° C., or greater than 70° C. The melting point ofan antibody can be measured using differential scanning calorimetry(Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) ImmunolLett 68:47-52) or circular dichroism (Murray et al. (2002) J. ChromatogrSci 40:343-9).

In another embodiment, the antibody is characterized by its resistanceto rapid degradation. Degradation of an antibody can be measured usingcapillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes DE (1995) Anal Chem 67:3626-32).

In another embodiment, the antibody exhibits minimal aggregationeffects, e.g., aggregation of 25% or less, such as 20% or less, 15% orless, 10% or less, 5% or less, or 4% or less. Aggregation can lead tothe triggering of an unwanted immune response and/or altered orunfavorable pharmacokinetic properties, Aggregation can be measured byseveral techniques, including size-exclusion column (SEC), highperformance liquid chromatography (HPLC), and light scattering.

In another embodiment, the antibody further exhibits at least one of thefollowing properties:

(a) binding to monkey LAG-3;

(b) lack of binding to mouse LAG-3;

(c) inhibition of binding of LAG-3 to major histocompatibility (MHC)class II molecules; and

(d) stimulation of immune responses, particularly antigen-specific Tcell responses.

Preferably, the antibody exhibits at least two of properties (a), (b),(c) and (d). More preferably, the antibody exhibits at least three ofproperties (a), (b), (c) and (d). Even more preferably, the antibodyexhibits all four of properties (a), (b), (c) and (d).

In another embodiment, the antibody stimulates an antigen-specific Tcell response, such as interleukin-2 (IL-2) production in anantigen-specific T cell response. In other embodiments, the antibodystimulates an immune response, such as an anti-tumor response (e.g.,inhibition of tumor growth in an in vivo tumor graft model) or anautoimmune response (e.g., development of diabetes in NOD mice).

In another embodiment, the antibody binds an epitope of human LAG-3comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 21). In anotherembodiment, the antibody binds an epitope of human LAG-3 comprising theamino acid sequence HPAAPSSW (SEQ ID NO: 22) or PAAPSSWG (SEQ ID NO:23).

In other embodiments, the antibody stains pituitary tissue byimmunohistochemistry, or does not stain pituitary tissue byimmunohistochemistry.

Antibodies of the invention can be full-length antibodies, for example,of an IgG1, IgG2 or IgG4 isotype, optionally with a serine to prolinemutation in the heavy chain constant region hinge region (at a positioncorresponding to position 241 as described in Angal et al. (1993) Mol.Immunol. 30:105-108), such that inter-heavy chain disulfide bridgeheterogeneity is reduced or abolished. In one aspect, the constantregion isotype is IgG4 with a mutation at amino acid residues 228, e.g.,S228P. Alternatively, the antibodies can be antibody fragments, such asFab, Fab′ or Fab′2 fragments, or single chain antibodies.

In another aspect of the invention, the antibody (or antigen-bindingportion thereof) is part of an immunoconjugate which includes atherapeutic agent, e.g., a cytotoxin or a radioactive isotope, linked tothe antibody. In another aspect, the antibody is part of a bispecificmolecule which includes a second functional moiety (e.g., a secondantibody) having a different binding specificity than said antibody, orantigen binding portion thereof.

Compositions comprising antibodies, or antigen-binding portions thereof,immunoconjugates or bispecific molecules of the invention, optionallyformulated in a pharmaceutically acceptable carrier, also are provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions (e.g., variable regions and/or CDRs) thereof, of the inventionalso are provided, as well as expression vectors comprising such nucleicacids and host cells comprising such expression vectors. Methods forpreparing anti-LAG-3 antibodies using the host cells comprising suchexpression vectors also are provided, and can include the steps of (i)expressing the antibody in the host cell and (ii) isolating the antibodyfrom the host cell.

In another aspect, the invention provides methods of stimulating immuneresponses using anti-LAG-3 antibodies of the invention. In oneembodiment, the method involves stimulating an antigen-specific T cellresponse by contacting T cells with an antibody of the invention, suchthat an antigen-specific T cell response is stimulated. In a preferredembodiment, interleukin-2 production by the antigen-specific T cell isstimulated. In another embodiment, the subject is a tumor-bearingsubject and an immune response against the tumor is stimulated. Inanother embodiment, the subject is a virus-bearing subject and an immuneresponse against the virus is stimulated.

In yet another embodiment, the invention provides a method forinhibiting growth of tumor cells in a subject comprising administeringto the subject an antibody, or antigen-binding portion thereof, of theinvention, such that growth of the tumor is inhibited in the subject. Instill another embodiment, the invention provides a method for treatingviral infection in a subject comprising administering to the subject anantibody, or antigen-binding portion thereof, of the invention such thatthe viral infection is treated in the subject. In another embodiment,these methods comprise administering a composition, bispecific, orimmunoconjugate of the invention.

In yet another embodiment, the invention provides a method forstimulating an immune response in a subject comprising administering tothe subject an antibody, or antigen-binding portion thereof, of theinvention and at least one additional immunostimulatory antibody, suchas an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4antibody, such that an immune response is stimulated in the subject, forexample to inhibit tumor growth or to stimulate an anti-viral response.In one embodiment, the additional immunostimulatory antibody is ananti-PD-1 antibody.

In another embodiment, the additional immunostimulatory agent is ananti-PD-L1 antibody. In yet another embodiment, the additionalimmunostimulatory agent is an anti-CTLA-4 antibody. In yet anotherembodiment, an antibody, or antigen-binding portion thereof, of theinvention is administered with a cytokine (e.g., IL-2 and/or IL-21), ora costimulatory antibody (e.g., an anti-CD137 and/or anti-GITRantibody). The antibodies can be, for example, human, chimeric orhumanized antibodies.

In another aspect, the invention provides anti-LAG-3 antibodies andcompositions of the invention for use in the foregoing methods, or forthe manufacture of a medicament for use in the foregoing methods (e.g.,for treatment).

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting. The contents of all references, Genbankentries, patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 1) and amino acidsequence (SEQ ID NO: 2) of the heavy chain variable region of the 25F7human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 6)and CDR3 (SEQ ID NO: 7) regions are delineated and the V, D and Jgermline derivations are indicated. The CDR regions are delineated usingthe Kabat system (Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 3) and amino acidsequence (SEQ ID NO: 4) of the kappa light chain variable region of the25F7 human monoclonal antibody. The CDR1 (SEQ ID NO: 8), CDR2 (SEQ IDNO: 9) and CDR3 (SEQ ID NO: 10) regions are delineated and the V and Jgermline derivations are indicated. The full-length heavy and lightchain amino acid sequences antibody 25F7 are shown in SEQ ID NOs: 32 and34, respectively.

FIG. 2A shows the amino acid sequence (SEQ ID NO: 12) of the heavy chainvariable region of the LAG3.5 monoclonal antibody. The CDR1 (SEQ ID NO:15), CDR2 (SEQ ID NO: 16) and CDR3 (SEQ ID NO: 17) regions aredelineated. The full-length heavy and light chain amino acid sequencesantibody LAG3.5 are shown in SEQ ID NOs: 35 and 37, respectively.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 13) and amino acidsequence (SEQ ID NO: 14) of the kappa light chain variable region of theLAG3.5 monoclonal antibody. The CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO: 20) regions are delineated.

FIG. 3 shows the amino acid sequences of the CDR2 heavy chain variableregion sequences of the LAG-3 variants LAG3.5 (SEQ ID NO: 42), LAG3.6(SEQ ID NO: 43), LAG3.7 (SEQ ID NO: 44), and LAG3.8 (SEQ ID NO: 45),compared to the amino acid sequence of the CDR2 heavy chain variableregion sequence of antibody 25F7 (LAG3.1) (SEQ ID NO: 41) andcorresponding human germline sequence (SEQ ID NO: 27). The CDR2 heavychain variable region of LAG3.5 differs from the CDR2 heavy chainvariable region of 25F7 by arginine (R) at position 54 (versusasparagine (N)) and serine (S) at position 56 (versus asparagines (N)).The remaining CDRs of LAG3.5 anf 25F7 are identical. FIG. 3 alsodiscloses SEQ ID NO: 40.

FIGS. 4A and 4B are graphs showing the binding activity (EC₅₀ andaffinity, respectively) of antibodies LAG3.1 (25F7), LAG3.2, LAG3.5,LAG3.6, LAG3.7, and LAG3.8 to activated human CD4+ T cells. FIG. 4Bdiscloses SEQ ID NOS 41, 42, 45, 44, and 43, respectively, in order ofappearance.

FIGS. 5A, B, C, D, and E are graphs showing thermal melting curves(i.e., thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6,LAG3.7, and LAG3.8, respectively.

FIGS. 6A, B, C, D, and E are graphs showing thermal reversibility curves(i.e., thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6,LAG3.7, and LAG3.8, respectively.

FIG. 7 is a graph, showing the binding activity of antibodies LAG3.1(25F7) and LAG3.5 to activated human CD4+ T cells and antigen binding(Biacore).

FIG. 8 shows the results of peptide mapping using mass-spectrometry(chemical modifications/molecular stability) for antibodies LAG3.1(25F7) and LAG3.5 reflecting deamidation and isomerization afterincubating for 5 days under accelerated stress conditions as describedherein. FIG. 8 discloses SEQ ID NOS 46-52, respectively, in order ofappearance.

FIG. 9 is a graph comparing the hydrophilicity profiles of antibodiesLAG3.1 (25F7) and LAG3.5.

FIGS. 10 A, B, C, and D are graphs comparing the affinity and physicalstability (i.e., thermal and chemical stability) of antibodies LAG3.1and LAG3.5 at 4° C. and 40° C., i.e., both accelerated stress conditionsand “real-time” stability studies, as described herein.

FIGS. 11A and B are graphs comparing the percent modification of theamino acid sequences of antibodies LAG3.1 and LAG3.5 at 4° C. and 40°C..

DETAILED DESCRIPTION OF THE INVENTION

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “25F7,” “antibody 25F7,” “antibody LAG3.1,” and “LAG3.1” referto the anti-human LAG-3 antibody described in US2011/0150892 A1. Thenucleotide sequence (SEQ ID NO: 1) encoding the heavy chain variableregion of 25F7 (LAG3.1) and the corresponding amino acid sequence (SEQID NO: 2) is shown in FIG. 1A (with CDR sequences designated as SEQ IDNOs: 4, 5, and 7, respectively). The nucleotide sequence (SEQ ID NO: 3)encoding the light chain variable region of 25F7 (LAG3.1) and thecorresponding amino acid sequence (SEQ ID NO: 4) is shown in FIG. 1B(with CDR sequences designated as SEQ ID NOs: 8, 9, and 10,respectively).

The term “LAG-3” refers to Lymphocyte Activation Gene-3. The term“LAG-3” includes variants, isoforms, homologs, orthologs and paralogs.For example, antibodies specific for a human LAG-3 protein may, incertain cases, cross-react with a LAG-3 protein from a species otherthan human. In other embodiments, the antibodies specific for a humanLAG-3 protein may be completely specific for the human LAG-3 protein andmay not exhibit species or other types of cross-reactivity, or maycross-react with LAG-3 from certain other species but not all otherspecies (e.g., cross-react with monkey LAG-3 but not mouse LAG-3). Theterm “human LAG-3” refers to human sequence LAG-3, such as the completeamino acid sequence of human LAG-3 having Genbank Accession No. NP002277 (SEQ ID NO: 29). The term “mouse LAG-3” refers to mouse sequenceLAG-3, such as the complete amino acid sequence of mouse LAG-3 havingGenbank Accession No. NP_(—)032505. LAG-3 is also known in the art as,for example, CD223. The human LAG-3 sequence may differ from human LAG-3of Genbank Accession No. NP_(—)002277 by having, e.g., conservedmutations or mutations in non-conserved regions and the LAG-3 hassubstantially the same biological function as the human LAG-3 of GenbankAccession No. NP_(—)002277. For example, a biological function of humanLAG-3 is having an epitope in the extracellular domain of LAG-3 that isspecifically bound by an antibody of the instant disclosure or abiological function of human LAG-3 is binding to MHC Class II molecules.

The term “monkey LAG-3” is intended to encompass LAG-3 proteinsexpressed by Old World and New World monkeys, including but not limitedto cynomolgus monkey LAG-3 and rhesus monkey LAG-3. A representativeamino acid sequence for monkey LAG-3 is the rhesus monkey LAG-3 aminoacid sequence which is also deposited as Genbank Accession No.XM_(—)001108923. Another representative amino acid sequence for monkeyLAG-3 is the alternative rhesus monkey sequence of clone pa23-5 asdescribed in US 2011/0150892 A1. This alternative rhesus sequenceexhibits a single amino acid difference, at position 419, as compared tothe Genbank-deposited sequence.

A particular human LAG-3 sequence will generally be at least 90%identical in amino acid sequence to human LAG-3 of Genbank Accession No.NP_(—)002277 and contains amino acid residues that identify the aminoacid sequence as being human when compared to LAG-3 amino acid sequencesof other species (e.g., murine). In certain cases, a human LAG-3 can beat least 95%, or even at least 96%, 97%, 98%, or 99% identical in aminoacid sequence to LAG-3 of Genbank Accession No. NP_(—)002277. In certainembodiments, a human LAG-3 sequence will display no more than 10 aminoacid differences from the LAG-3 sequence of Genbank Accession No.NP_(—)002277. In certain embodiments, the human LAG-3 can display nomore than 5, or even no more than 4, 3, 2, or 1 amino acid differencefrom the LAG-3 sequence of Genbank Accession No. NP_(—)002277. Percentidentity can be determined as described herein.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

An “antigen-specific T cell response” refers to responses by a T cellthat result from stimulation of the T cell with the antigen for whichthe T cell is specific. Non-limiting examples of responses by a T cellupon antigen-specific stimulation include proliferation and cytokineproduction (e.g., IL-2 production).

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. Whole antibodies are glycoproteins comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies canmediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., a LAG-3 protein). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(H) and C_(H)1 domains; (v) a Fv fragment consistingof the V_(L) and V_(H) domains of a single arm of an antibody, (vi) adAb fragment (Ward et al., (1989) Nature 341:544-546), which consists ofa V_(H) domain; (vii) an isolated complementarity determining region(CDR); and (viii) a nanobody, a heavy chain variable region containing asingle variable domain and two constant domains. Furthermore, althoughthe two domains of the Fv fragment, V_(L) and V_(H), are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the V_(L) and V_(H) regions pair to form monovalent molecules(known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds a LAG-3 protein is substantially free of antibodies thatspecifically bind antigens other than LAG-3 proteins). An isolatedantibody that specifically binds a human LAG-3 protein may, however,have cross-reactivity to other antigens, such as LAG-3 proteins fromother species. Moreover, an isolated antibody can be substantially freeof other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention can include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity, which have variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic nonhuman animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

The term “isotype” refers to the antibody class (e.g., IgM or IgG1) thatis encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications can be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds human LAG-3” isintended to refer to an antibody that binds to human LAG-3 protein (andpossibly a LAG-3 protein from one or more non-human species) but doesnot substantially bind to non-LAG-3 proteins. Preferably, the antibodybinds to a human LAG-3 protein with “high affinity”, namely with a K_(D)of 1×10⁻⁷ M or less, more preferably 1×10⁻⁸ M or less, more preferably5×10⁻⁹ M or less, more preferably 1×10⁻⁹ M or less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D),” as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a). (i.e., K_(d)/K_(a)) and is expressed as a molar concentration(M). K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

The term “high affinity” for an IgG antibody refers to an antibodyhaving a K_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less,even more preferably 1×10⁻⁸ M or less, even more preferably 5×10⁻⁹ M orless and even more preferably 1×10⁻⁹ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁶ M or less, more preferably 10⁻⁷ M orless, even more preferably 10⁻⁸ M or less.

The term “deamidation” refers to a chemical degredative process thatspontaneously occurs in proteins (e.g., antibodies). Deamidation removesan amide functional group from an amino acid residue, such as asparagineand glutamine, thus damaging its amide-containing side chains.Specifically, the side chain of an asparagine attacks the adjacentpeptide group, forming a symmetric succinimide intermediate. Thesymmetry of the intermediate results in two hydrolysis products, eitheraspartate or isoaspartate. A similar reaction can also occur inaspartate side chains, yielding a partial conversion to isoaspartate. Inthe case of glutamine, the rate of deamidation is generally ten foldless than asparagine, however, the mechanism is essentially the same,requiring only water molecules to proceed.

The term “subject” includes any human or nonhuman animal. The term“nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles, although mammals arepreferred, such as non-human primates, sheep, dogs, cats, cows andhorses.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-LAG-3 Antibodies Having Increased Stability and AdvantageousFunctional Properties

Antibodies of the invention specifically bind to human LAG-3 and haveoptimized stability compared to previously described anti-LAG-3antibodies, particularly compared to antibody 25F7 (LAG3.1). Thisoptimization includes reduced deamidation (e.g., increased chemicalstability) and increased thermal refolding (e.g., increased physicalstability), while still retaining high affinity binding to human LAG-3.

Methods for identifying deamidation sites are known in the art (see,e.g., ion exchange, reversed phase, and hydrophobic interactionchromatography, and peptide mapping of proteolytic digests (LC-MS)).Suitable assays for measuring physical stability include, e.g., analysisof melting points and/or refolding of antibody structure followingdenaturation (e.g., percent reversibility as described, e.g., in Example3, Section 3).

Binding to human LAG-3 can be assessed using one or more techniques alsowell established in the art. For example, an antibody can be tested by aflow cytometry assay in which the antibody is reacted with a cell linethat expresses human LAG-3, such as CHO cells that have been transfectedto express LAG-3 (e.g., human LAG-3, or monkey LAG-3 (e.g., rhesus orcynomolgus monkey) or mouse LAG-3) on their cell surface. Other suitablecells for use in flow cytometry assays include anti-CD3-stimulated CD4⁺activated T cells, which express native LAG-3. Additionally oralternatively, binding of the antibody, including the binding kinetics(e.g., K_(D) value), can be tested in BIAcore assays. Still othersuitable binding assays include ELISA assays, for example, using arecombinant LAG-3 protein.

Antibodies of the invention preferably bind to human LAG-3 protein witha K_(D) of 1×10⁻⁷ M or less, and more preferably 1×10⁻⁸ M or less,5×10⁻⁹ M or less, or 1×10⁻⁹ M or less.

Typically, the antibody binds to LAG-3 in lymphoid tissues, such astonsil, spleen or thymus, which can be detected by immunohistochemistry.In one embodiment, the antibody stains pituitary tissue (e.g., areretained in the pituitary) as measured by immunohistochemistry. Inanother embodiment, the antibody does not stain pituitary tissue (i.e.,is not retained in the pituitary) as measured by immunohistochemistry.

Additional functional properties include cross-reactivity with LAG-3from other species. For example, the antibody can bind to monkey LAG-3(e.g., cynomolgus monkey, rhesus monkey), but not substantially bind toLAG-3 from mouse LAG-3. Preferably, an antibody of the invention bindsto human LAG-3 with high affinity.

Other functional properties include the ability of the antibody tostimulate an immune response, such as an antigen-specific T cellresponse. This can be tested, for example, by assessing the ability ofthe antibody to stimulate interleukin-2 (IL-2) production in anantigen-specific T cell response. In certain embodiments, the antibodybinds to human LAG-3 and stimulates an antigen-specific T cell response.In other embodiments, the antibody binds to human LAG-3 but does notstimulate an antigen-specific T cell response. Other means forevaluating the capacity of the antibody to stimulate an immune responseinclude testing its ability to inhibit tumor growth, such as in an invivo tumor graft model (see, e.g., Example 6) or the ability tostimulate an autoimmune response, such as the ability to promote thedevelopment of an autoimmune disease in an autoimmune model, e.g., theability to promote the development of diabetes in the NOD mouse model.

Preferred antibodies of the invention are human monoclonal antibodies.Additionally or alternatively, the antibodies can be, for example,chimeric or humanized monoclonal antibodies.

Monoclonal Antibody LAG3.5

A preferred antibody of the invention is the human monoclonal antibody,LAG3.5, structurally and chemically characterized as described below andin the following Examples. The V_(H) amino acid sequence of LAG3.5 isshown in SEQ ID NO: 12 (FIG. 2A). The V_(L) amino acid sequence ofLAG3.5 is shown in SEQ ID NO: 14 (FIG. 2B).

The V_(H) and V_(L) sequences (or CDR sequences) of other anti-LAG-3antibodies which bind human LAG-3 can be “mixed and matched” with theV_(H) and V_(L) sequences (or CDR sequences) of antibody LAG3.5.Preferably, when V_(H) and V_(L) chains (or the CDRs within such chains)are mixed and matched, a V_(H) sequence from a particular V_(H)/V_(L)pairing is replaced with a structurally similar V_(H) sequence.Likewise, preferably a V_(L) sequence from a particular V_(H)N_(L)pairing is replaced with a structurally similar V_(L) sequence.

Accordingly, in one embodiment, antibodies of the invention, or antigenbinding portions thereof, comprise:

(a) a heavy chain variable region comprising amino acid sequence SEQ IDNO: 12 (i.e., the V_(H) of LAG3.5); and

(b) a light chain variable region comprising amino acid sequence SEQ IDNO: 14 (i.e., the V_(L) of LAG3.5) or the V_(L) of another anti-LAG3antibody (i.e., which differs from LAG3.5);

wherein the antibody specifically binds human LAG-3.

In another embodiment, antibodies of the invention, or antigen bindingportions thereof, comprise:

(a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable regioncomprising amino acid sequence SEQ ID NO: 12 (i.e., the CDR sequences ofLAG3.5, SEQ ID NOs:15, 16, and 17, respectively); and

(b) the CDR1, CDR2, and CDR3 regions of the light chain variable regioncomprising amino acid sequence SEQ ID NO: 14 (i.e., the CDR sequences ofLAG3.5, SEQ ID NOs:18, 19, and 20, respectively) or the CDRs of anotheranti-LAG3 antibody (i.e., which differs from LAG3.5);

wherein the antibody specifically binds human LAG-3.

In yet another embodiment, the antibody, or antigen binding portionthereof, includes the heavy chain variable CDR2 region of LAG3.5combined with CDRs of other antibodies which bind human LAG-3, e.g., aCDR1 and/or CDR3 from the heavy chain variable region, and/or a CDR1,CDR2, and/or CDR3 from the light chain variable region of a differentanti-LAG-3 antibody.

In addition, it is well known in the art that the CDR3 domain,independently from the CDR1 and/or CDR2 domain(s), alone can determinethe binding specificity of an antibody for a cognate antigen and thatmultiple antibodies can predictably be generated having the same bindingspecificity based on a common CDR3 sequence. See, e.g., Klimka et al.,British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol.296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. U.S.A.95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162(1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533(1995); Ditzel et al., J. Immunol. 157:739-749 (1996); Berezov et al.,BIAjournal 8:Scientific Review 8 (2001); Igarashi et al., J. Biochem(Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998);Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993); Polymenisand Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity13:37-45 (2000). See also, U.S. Pat. Nos. 6,951,646; 6,914,128;6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and5,760,185. Each of these references is hereby incorporated by referencein its entirety.

Accordingly, in another embodiment, antibodies of the invention includethe CDR2 of the heavy chain variable region of LAG3.5 and at least theCDR3 of the heavy and/or light chain variable region of LAG3.5 (SEQ IDNOs: 17 and/or 20), or the CDR3 of the heavy and/or light chain variableregion of another LAG-3 antibody, wherein the antibody is capable ofspecifically binding to human LAG-3. These antibodies preferably (a)compete for binding with; (b) retain the functional characteristics; (c)bind to the same epitope; and/or (d) have a similar binding affinity asLAG3.5. In yet another embodiment, the antibodies further may includethe CDR2 of the light chain variable region of LAG3.5 (SEQ ID NOs: 17and/or 20), or the CDR2 of the light chain variable region of anotherLAG-3 antibody, wherein the antibody is capable of specifically bindingto human LAG-3. In another embodiment, the antibodies of the inventionfurther may include the CDR1 of the heavy and/or light chain variableregion of LAG3.5 (SEQ ID NOs: 17 and/or 20), or the CDR1 of the heavyand/or light chain variable region of another LAG-3 antibody, whereinthe antibody is capable of specifically binding to human LAG-3.

Conservative Modifications

In another embodiment, antibodies of the invention comprise a heavyand/or light chain variable region sequences of CDR1, CDR2 and CDR3sequences which differ from those of LAG3.5 by one or more conservativemodifications. In a preferred embodiment, however, residues 54 and 56 ofthe V_(H) CDR2 remain as arginine and serine, respectively (i.e., arenot mutated). It is understood in the art that certain conservativesequence modification can be made which do not remove antigen binding.See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al.(1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem.272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley andO'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int.Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.Accordingly, in one embodiment, the antibody comprises a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and/or a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences,wherein:

(a) the heavy chain variable region CDR1 sequence comprises SEQ ID NO:15, and/or conservative modifications thereof, except at positions 54and 56; and/or

(b) the heavy chain variable region CDR3 sequence comprises SEQ ID NO:17, and conservative modifications thereof; and/or

(c) the light chain variable region CDR1, and/or CDR2, and/or CDR3sequences comprise SEQ ID NO: 18, and/or, SEQ ID NO: 19, and/or SEQ IDNO: 20, and/or conservative modifications thereof; and

(d) the antibody specifically binds human LAG-3.

Additionally or alternatively, the antibody can possess one or more ofthe following functional properties described above, such as highaffinity binding to human LAG-3, binding to monkey LAG-3, lack ofbinding to mouse LAG-3, the ability to inhibit binding of LAG-3 to MHCClass II molecules and/or the ability to stimulate antigen-specific Tcell responses.

In various embodiments, the antibody can be, for example, a human,humanized or chimeric antibody

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth above) using the functionalassays described herein.

Engineered and Modified Antibodies

Antibodies of the invention can be prepared using an antibody having oneor more of the V_(H) and/or V_(L) sequences of LAG3.5 as startingmaterial to engineer a modified antibody. An antibody can be engineeredby modifying one or more residues within one or both variable regions(i.e., V_(H) and/or V_(L)), for example within one or more CDR regionsand/or within one or more framework regions. Additionally oralternatively, an antibody can be engineered by modifying residueswithin the constant region(s), for example to alter the effectorfunction(s) of the antibody.

In certain embodiments, CDR grafting can be used to engineer variableregions of antibodies. Antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann et al. (1998) Nature332:323-327; Jones et al. (1986) Nature 321:522-525; Queen et al. (1989)Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. Nos. 5,225,539;5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising SEQ ID NOs: 15, 16, 17, respectively, and/or a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences comprising SEQID NOs: 18, 19, 20, respectively (i.e., the CDRs of LAG3.5). While theseantibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibody LAG3.5, they can contain differing framework sequences.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat et al. (1991), cited supra; Tomlinson et al. (1992)“The Repertoire of Human Germline V_(H) Sequences Reveals about FiftyGroups of V_(H) Segments with Different Hypervariable Loops” J. Mol.Biol. 227:776-798; and Cox et al. (1994) “A Directory of Human Germ-lineV_(H) Segments Reveals a Strong Bias in their Usage” Eur. J. Immunol.24:827-836; the contents of each of which are expressly incorporatedherein by reference. As another example, the germline DNA sequences forhuman heavy and light chain variable region genes can be found in theGenbank database. For example, the following heavy chain germlinesequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank Accession Nos.: 1-69 (NG_(—)0010109, NT_(—)024637 &BC070333), 3-33 (NG_(—)0010109 & NT_(—)024637) and 3-7 (NG_(—)0010109 &NT_(—)024637). As another example, the following heavy chain germlinesequences found in the HCo12 HuMAb mouse are available in theaccompanying Genbank Accession Nos.: 1-69 (NG_(—)0010109, NT_(—)024637 &BC070333), 5-51 (NG_(—)0010109 & NT_(—)024637), 4-34 (NG_(—)0010109 &NT_(—)024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997), supra), which is wellknown to those skilled in the art.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the V_(H) 4-34framework sequences and/or the V_(K) L6 framework sequences used bypreferred monoclonal antibodies of the invention. The V_(H) CDR1, CDR2,and CDR3 sequences, and the V_(K) CDR1, CDR2, and CDR3 sequences, can begrafted onto framework regions that have the identical sequence as thatfound in the germline immunoglobulin gene from which the frameworksequence derive, or the CDR sequences can be grafted onto frameworkregions that contain one or more mutations as compared to the germlinesequences. For example, it has been found that in certain instances itis beneficial to mutate residues within the framework regions tomaintain or enhance the antigen binding ability of the antibody (seee.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations can be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the invention provides isolatedanti-LAG-3 monoclonal antibodies, or antigen binding portions thereof,comprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising SEQ ID NO: 15, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NO: 15; (b) a V_(H) CDR2 regioncomprising SEQ ID NO: 16, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NO: 16 (preferably wherein positions 54 and 56 arethe same as in SEQ ID NO:16); (c) a V_(H) CDR3 region comprising SEQ IDNO: 17, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNO: 17; (d) a V_(L) CDR1 region comprising SEQ ID NO: 18, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NO: 18; (e)a V_(L) CDR2 region comprising SEQ ID NO: 19, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NO: 19; and (f) a V_(L) CDR3 regioncomprising SEQ ID NO: 20, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NO: 20.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation cancontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention can be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention can bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In a preferred embodiment, the antibody is an IgG4 isotype antibodycomprising a Serine to Proline mutation at a position corresponding toposition 228 (S228P; EU index) in the hinge region of the heavy chainconstant region. This mutation has been reported to abolish theheterogeneity of inter-heavy chain disulfide bridges in the hinge region(Angal et al. supra; position 241 is based on the Kabat numberingsystem).

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, toincrease the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fey receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutationsat positions 256, 290, 298, 333, 334 and 339 were shown to improvebinding to FcγRIII. Additionally, the following combination mutants wereshown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224Aand S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. See, e.g., U.S. Pat.Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8(α(1,6)-fucosyltransferase), such that antibodies expressed in theMs704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.The Ms704, Ms705, and Ms709 FUT8^(−/−) cell lines were created by thetargeted disruption of the FUT8 gene in CHO/DG44 cells using tworeplacement vectors (see U.S. Patent Publication No. 20040110704 andYamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 describes a cell line with a functionallydisrupted FUT8 gene, which encodes a fucosyl transferase, such thatantibodies expressed in such a cell line exhibit hypofucosylation byreducing or eliminating the α-1,6 bond-related enzyme. EP 1,176,195 alsodescribes cell lines which have a low enzyme activity for adding fucoseto the N-acetylglucosamine that binds to the Fc region of the antibodyor does not have the enzyme activity, for example the rat myeloma cellline YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes avariant CHO cell line, Lec13 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with amodified glycosylation profile can also be produced in chicken eggs, asdescribed in PCT Publication WO 06/089231. Alternatively, antibodieswith a modified glycosylation profile can be produced in plant cells,such as Lemna. Methods for production of antibodies in a plant systemare disclosed in the U.S. patent application corresponding to Alston &Bird LLP attorney docket No. 040989/314911, filed on Aug. 11, 2006. PCTPublication WO 99/54342 describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody can be cleaved offusing a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removesfucosyl residues from antibodies (Tarentino et al. (1975) Biochem.14:5516-23).

Another modification of the antibodies herein that is contemplated bythis disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See, e.g., EPO 154 316 and EP 0 401384.

Antibody Physical Properties

Antibodies of the invention can be characterized by their variousphysical properties, to detect and/or differentiate different classesthereof.

For example, antibodies can contain one or more glycosylation sites ineither the light or heavy chain variable region. Such glycosylationsites may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-LAG-3antibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

In a preferred embodiment, the antibodies do not contain asparagineisomerism sites. The deamidation of asparagine may occur on N-G or D-Gsequences and result in the creation of an isoaspartic acid residue thatintroduces a kink into the polypeptide chain and decreases its stability(isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-LAG-3 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Nucleic Acid Molecules Encoding Antibodies of the Invention

In another aspect, the invention provides nucleic acid molecules thatencode heavy and/or light chain variable regions, or CDRs, of theantibodies of the invention. The nucleic acids can be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form. A nucleic acid is “isolated” or “rendered substantially pure”when purified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See,Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology,Greene Publishing and Wiley Interscience, New York. A nucleic acid ofthe invention can be, e.g., DNA or RNA and may or may not containintronic sequences. In a preferred embodiment, the nucleic acid is acDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), a nucleic acid encoding such antibodies can be recoveredfrom the gene library.

Preferred nucleic acids molecules of the invention include thoseencoding the V_(H) and V_(L) sequences of LAG3.5 monoclonal antibody(SEQ ID NOs: 12 and 14, respectively) or the CDRs. Once DNA fragmentsencoding V_(H) and V_(L) segments are obtained, these DNA fragments canbe further manipulated by standard recombinant DNA techniques, forexample to convert the variable region genes to full-length antibodychain genes, to Fab fragment genes or to a scFv gene. In thesemanipulations, a V_(L)- or V_(H)-encoding DNA fragment is operativelylinked to another DNA fragment encoding another protein, such as anantibody constant region or a flexible linker. The term “operativelylinked”, as used in this context, is intended to mean that the two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat et al. (1991), supra) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the V_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabatet al., supra) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. In preferred embodiments, thelight chain constant region can be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃ (SEQ ID NO: 28), such thatthe V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) regions joined by theflexible linker (see e.g., Bird et al. (1988) Science 242:423-426;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCaffertyet al., (1990) Nature 348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be producedusing the well-known somatic cell hybridization (hybridoma) technique ofKohler and Milstein (1975) Nature 256: 495. Other embodiments forproducing monoclonal antibodies include viral or oncogenictransformation of B lymphocytes and phage display techniques. Chimericor humanized antibodies are also well known in the art. See e.g., U.S.Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and6,180,370, the contents of which are specifically incorporated herein byreference in their entirety.

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againsthuman LAG-3 can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as the HuMAb Mouse® and KM Mouse®, respectively, and arecollectively referred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg et al. (1994), supra; reviewed in Lonberg(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding andLonberg (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use ofthe HuMAb Mouse®, and the genomic modifications carried by such mice, isfurther described in Taylor et al. (1992) Nucleic Acids Research20:6287-6295; Chen et al. (1993) International Immunology 5: 647-656;Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi etal. (1993) Nature Genetics 4:117-123; Chen et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor et al.(1994) International Immunology 6: 579-591; and Fishwild et al. (1996)Nature Biotechnology 14: 845-851, the contents of all of which arehereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and5,545,807; PCT Publication Nos. WO 92/03918; WO 93/12227; WO 94/25585;WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424, the contents ofwhich are incorporated herein by reference in their entirety.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. This mouse isreferred to herein as a “KM Mouse®,” and is described in detail in PCTPublication WO 02/43478. A modified form of this mouse, which furthercomprises a homozygous disruption of the endogenous FcγRIIB receptorgene, is also described in PCT Publication WO 02/43478 and referred toherein as a “KM/FCGR2D mouse.” In addition, mice with either the HCo7 orHCo12 heavy chain transgenes or both can be used.

Additional transgenic animal embodiments include the Xenomouse (Abgenix,Inc., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and6,162,963). Further embodiments include “TC mice” (Tomizuka et al.(2000) Proc. Natl. Acad. Sci. USA 97:722-727) and cows carrying humanheavy and light chain transchromosomes (Kuroiwa et al. (2002) NatureBiotechnology 20:889-894; PCT Publication WO 02/092812). The contents ofthese patents and publications are specifically incorporated herein byreference in their entirety.

In one embodiment, human monoclonal antibodies of the invention areprepared using phage display methods for screening libraries of humanimmunoglobulin genes. See, e.g. U.S. Pat. Nos. 5,223,409; 5,403,484;5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793;6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081, the contentsof which are incorporated herein by reference in their entirety.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. See,e.g., U.S. Pat. Nos. 5,476,996 and 5,698,767, the contents of which areincorporated herein by reference in their entirety.

In another embodiment, human anti-LAG-3 antibodies are prepared usingphage display where the phages comprise nucleic acids encodingantibodies generated in transgenic animals previously immunized withLAG-3. In a preferred embodiment, the transgenic animal is a HuMab, KM,or Kirin mouse. See, e.g. U.S. Pat. No. 6,794,132, the contents of whichare incorporated herein by reference in its entirety.

Immunization of Human Ig Mice

In one embodiment of the invention, human Ig mice are immunized with apurified or enriched preparation of a LAG-3 antigen, recombinant LAG-3protein, or cells expressing a LAG-3 protein. See, e.g., Lonberg et al.(1994), supra; Fishwild et al. (1996), supra; PCT Publications WO98/24884 or WO 01/14424, the contents of which are incorporated hereinby reference in their entirety. In a preferred embodiment, 6-16 week oldmice are immunized with 5-50 μg of LAG-3 protein. Alternatively, aportion of LAG-3 fused to a non-LAG-3 polypeptide is used.

In one embodiment, the transgenic mice are immunized intraperitoneally(IP) or intravenously (IV) with LAG-3 antigen in complete Freund'sadjuvant, followed by subsequent IP or IV immunizations with antigen inincomplete Freund's adjuvant. In other embodiments, adjuvants other thanFreund's or whole cells in the absence of adjuvant are used. The plasmacan be screened by ELISA and cells from mice with sufficient titers ofanti-LAG-3 human immunoglobulin can be used for fusions.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. Generation of hybridomasis well-known in the art. See, e.g., Harlow and Lane (1988) Antibodies,A Laboratory Manual, Cold Spring Harbor Publications, New York.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNAencoding partial or full-length light and heavy chains obtained bystandard molecular biology techniques is inserted into one or moreexpression vectors such that the genes are operatively linked totranscriptional and translational regulatory sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodychain genes. Such regulatory sequences are described, e.g., in Goeddel(Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990)). Preferred regulatory sequences for mammalianhost cell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),adenovirus, (e.g., the adenovirus major late promoter (AdMLP) andpolyoma. Alternatively, nonviral regulatory sequences can be used, suchas the ubiquitin promoter or β-globin promoter. Still further,regulatory elements composed of sequences from different sources, suchas the SRα promoter system, which contains sequences from the SV40 earlypromoter and the long terminal repeat of human T cell leukemia virustype 1 (Takebe et al. (1988) Mol. Cell. Biol. 8:466-472). The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can beinserted into the same or separate expression vectors. In preferredembodiments, the variable regions are used to create full-lengthantibody genes of any antibody isotype by inserting them into expressionvectors already encoding heavy chain constant and light chain constantregions of the desired isotype such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector and theV_(L) segment is operatively linked to the C_(L) segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216; 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr⁻ CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Immunoconjugates

Antibodies of the invention can be conjugated to a therapeutic agent toform an immunoconjugate such as an antibody-drug conjugate (ADC).Suitable therapeutic agents include antimetabolites, alkylating agents,DNA minor groove binders, DNA intercalators, DNA crosslinkers, histonedeacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In the ADC, the antibody and therapeutic agent preferably areconjugated via a linker cleavable such as a peptidyl, disulfide, orhydrazone linker. More preferably, the linker is a peptidyl linker suchas Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ IDNO: 39), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys,Cit, Ser, or Glu. The ADCs can be prepared as described in U.S. Pat.Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910;WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO08/103,693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference.

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising one or more antibodies of the invention linked to at leastone other functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. Thus, as used herein, “bispecific molecule” includesmolecules that have three or more specificities. In a preferredembodiment, the bispecific molecule comprises a first bindingspecificity for LAG-3 and a second binding specificity for a triggeringmolecule that recruits cytotoxic effector cells that can kill a LAG-3expressing target cell. Examples of suitable triggering molecules areCD64, CD89, CD16, and CD3. See, e.g., Kufer et al., TRENDS inBiotechnology, 22 (5), 238-244 (2004).

In an embodiment, a bispecific molecule has, in addition to an anti-Fcbinding specificity and an anti-LAG-3 binding specificity, a thirdspecificity. The third specificity can be for an anti-enhancement factor(EF), e.g., a molecule that binds to a surface protein involved incytotoxic activity and thereby increases the immune response against thetarget cell. For example, the anti-enhancement factor can bind acytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) orother immune cell, resulting in an increased immune response against thetarget cell.

Bispecific molecules can come in many different formats and sizes. Atone end of the size spectrum, a bispecific molecule retains thetraditional antibody format, except that, instead of having two bindingarms of identical specificity, it has two binding arms each having adifferent specificity. At the other extreme are bispecific moleculesconsisting of two single-chain antibody fragments (scFv's) linked by apeptide chain, a so-called Bs(scFv)₂ construct. Intermediate-sizedbispecific molecules include two different F(ab) fragments linked by apeptidyl linker. Bispecific molecules of these and other formats can beprepared by genetic engineering, somatic hybridization, or chemicalmethods. See, e.g., Kufer et al, cited supra; Cao and Suresh,Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al.,Immunology Today, 21 (8), 391-397 (2000), and the references citedtherein.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising one or more antibodies of the present inventionformulated together with a pharmaceutically acceptable carrier. Thecomposition may optionally contain one or more additionalpharmaceutically active ingredients, such as another antibody or a drug.The pharmaceutical compositions of the invention also can beadministered in a combination therapy with, for example, anotherimmunostimulatory agent, anti-cancer agent, an anti-viral agent, or avaccine, such that the anti-LAG-3 antibody enhances the immune responseagainst the vaccine.

The pharmaceutical composition can comprise any number of excipients.Excipients that can be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound can be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” as used hereinmeans modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, e.g., intranasally, orally, vaginally, rectally,sublingually or topically.

The pharmaceutical compositions of the invention can includepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects.Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to highdrug concentration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 0.01% to about ninety-nine percent of active ingredient,preferably from about 0.1% to about 70%, most preferably from about 1%to about 30% of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus can beadministered, several divided doses can be administered over time or thedose can be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Alternatively,antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-LAG-3antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks. In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

A “therapeutically effective dosage” of an anti-LAG-3 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumor-bearing subjects, a“therapeutically effective dosage” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic compound can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human or can beanother mammal.

The pharmaceutical composition can be a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparati(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example, toensure that the therapeutic compounds of the invention cross theblood-brain barrier, they can be formulated in liposomes, which mayadditionally comprise targeting moieties to enhance selective transportto specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811;5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin.Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038; Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al.(1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al. (1995) Am.J. Physiol. 1233:134; Schreier et al. (1994) J. Biol. Chem. 269:9090;Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler(1994) Immunomethods 4:273.

Uses and Methods of the Invention

Antibodies (compositions, bispecifics, and immunoconjugates) of thepresent invention have numerous in vitro and in vivo utilitiesinvolving, for example, detection of LAG-3 or enhancement of immuneresponses by blockade of LAG-3. In a preferred embodiment, theantibodies are human antibodies. Such antibodies can be administered tocells in culture, in vitro or ex vivo, or to human subjects, e.g., invivo, to enhance immunity in a variety of situations. Accordingly, inone aspect, the invention provides a method of modifying an immuneresponse in a subject comprising administering to the subject theantibody, or antigen-binding portion thereof, of the invention such thatthe immune response in the subject is modified. Preferably, the responseis enhanced, stimulated or up-regulated.

Preferred subjects include human patients in need of enhancement of animmune response. The methods are particularly suitable for treatinghuman patients having a disorder that can be treated by augmenting animmune response (e.g., the T-cell mediated immune response). In aparticular embodiment, the methods are particularly suitable fortreatment of cancer in vivo. To achieve antigen-specific enhancement ofimmunity, the anti-LAG-3 antibodies can be administered together with anantigen of interest or the antigen may already be present in the subjectto be treated (e.g., a tumor-bearing or virus-bearing subject). Whenantibodies to LAG-3 are administered together with another agent, thetwo can be administered in either order or simultaneously.

The invention further provides methods for detecting the presence ofhuman LAG-3 antigen in a sample, or measuring the amount of human LAG-3antigen, comprising contacting the sample, and a control sample, with ahuman monoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to human LAG-3, under conditions that allow forformation of a complex between the antibody or portion thereof and humanLAG-3. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of human LAG-3 antigen in the sample. Moreover,the anti-LAG-3 antibodies of the invention can be used to purify humanLAG-3 via immunoaffinity purification.

Given the ability of anti-LAG-3 antibodies of the invention to inhibitthe binding of LAG-3 to MHC Class II molecules and to stimulateantigen-specific T cell responses, the invention also provides in vitroand in vivo methods of using the antibodies to stimulate, enhance orupregulate antigen-specific T cell responses. For example, the inventionprovides a method of stimulating an antigen-specific T cell responsecomprising contacting said T cell with an antibody of the invention,such that an antigen-specific T cell response is stimulated. Anysuitable indicator of an antigen-specific T cell response can be used tomeasure the antigen-specific T cell response.

Non-limiting examples of such suitable indicators include increased Tcell proliferation in the presence of the antibody and/or increasecytokine production in the presence of the antibody. In a preferredembodiment, interleukin-2 production by the antigen-specific T cell isstimulated.

The invention also provides method for stimulating an immune response(e.g., an antigen-specific T cell response) in a subject comprisingadministering an antibody of the invention to the subject such that animmune response (e.g., an antigen-specific T cell response) in thesubject is stimulated. In a preferred embodiment, the subject is atumor-bearing subject and an immune response against the tumor isstimulated. In another preferred embodiment, the subject is avirus-bearing subject and an immune response against the virus isstimulated.

In another embodiment, the invention provides methods for inhibitinggrowth of tumor cells in a subject comprising administering to thesubject an antibody of the invention such that growth of the tumor isinhibited in the subject. In yet another embodiment, the inventionprovides methods for treating a viral infection in a subject comprisingadministering to the subject an antibody of the invention such that theviral infection is treated in the subject.

These and other methods of the invention are discussed in further detailbelow.

Cancer

Blockade of LAG-3 by antibodies can enhance the immune response tocancerous cells in the patient. In one aspect, the present inventionrelates to treatment of a subject in vivo using an anti-LAG-3 antibodysuch that growth of cancerous tumors is inhibited. An anti-LAG-3antibody can be used alone to inhibit the growth of cancerous tumors.Alternatively, an anti-LAG-3 antibody can be used in conjunction withother immunogenic agents, standard cancer treatments, or otherantibodies, as described below.

Accordingly, in one embodiment, the invention provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-LAG-3antibody, or antigen-binding portion thereof. Preferably, the antibodyis a human anti-LAG-3 antibody (such as any of the human anti-humanLAG-3 antibodies described herein). Additionally or alternatively, theantibody can be a chimeric or humanized anti-LAG-3 antibody.

Preferred cancers whose growth may be inhibited using the antibodies ofthe invention include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment includemelanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clearcell carcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), breast cancer, colon cancer and lung cancer (e.g.non-small cell lung cancer). Additionally, the invention includesrefractory or recurrent malignancies whose growth may be inhibited usingthe antibodies of the invention.

Examples of other cancers that can be treated using the methods of theinvention include bone cancer, pancreatic cancer, skin cancer, cancer ofthe head or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present invention is also useful fortreatment of metastatic cancers, especially metastatic cancers thatexpress PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144).

Optionally, antibodies to LAG-3 can be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By raising the threshold of T cell activation by LAG-3blockade, the tumor responses in the host can be activated.

LAG-3 blockade is likely to be more effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principlesand Practice of Oncology, Fifth Edition). In one of these strategies, avaccine is prepared using autologous or allogeneic tumor cells. Thesecellular vaccines have been shown to be most effective when the tumorcells are transduced to express GM-CSF. GM-CSF has been shown to be apotent activator of antigen presentation for tumor vaccination (Dranoffet al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. LAG-3 blockade can be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). (These somatic tissues may be protected from immuneattack by various means). Tumor antigen can also be “neo-antigens”expressed in cancer cells because of somatic mutations that alterprotein sequence or create fusion proteins between two unrelatedsequences (i.e., bcr-abl in the Philadelphia chromosome), or idiotypefrom B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with LAG-3blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withLAG-3 blockade to activate more potent anti-tumor responses.

LAG-3 blockade can also be combined with standard cancer treatments.LAG-3 blockade can be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is ananti-LAG-3 antibody in combination with decarbazine for the treatment ofmelanoma. Another example of such a combination is an anti-LAG-3antibody in combination with interleukin-2 (IL-2) for the treatment ofmelanoma. The scientific rationale behind the combined use of LAG-3blockade and chemotherapy is that cell death, that is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withLAG-3 blockade through cell death are radiation, surgery, and hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors can also be combined with LAG-3blockade. Inhibition of angiogenesis leads to tumor cell death which mayfeed tumor antigen into host antigen presentation pathways.

LAG-3 blocking antibodies can also be used in combination withbispecific antibodies that target Fcα or Fcγ receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would be augmentedby the use of LAG-3 blockade. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-LAG-3 to counteract the effects of the immunosuppressive agent andfavor tumor immune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-LAG-3. These include molecules on the surfaceof dendritic cells which activate DC function and antigen presentation.Anti-CD40 antibodies are able to substitute effectively for T cellhelper activity (Ridge et al. (1998) Nature 393: 474-478) and can beused in conjunction with LAG-3 antibodies (Ito et al. (2000)Immunobiology 201 (5) 527-40). Activating antibodies to T cellcostimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097),OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero etal. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al.(1999) Nature 397: 262-266) may also provide for increased levels of Tcell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. LAG-3 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-LAG-3 antibodies can increase the frequency andactivity of the adoptively transferred T cells.

Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. Accordingly, another aspectof the invention provides a method of treating an infectious disease ina subject comprising administering to the subject an anti-LAG-3antibody, or antigen-binding portion thereof, such that the subject istreated for the infectious disease. Preferably, the antibody is a humananti-human LAG-3 antibody (such as any of the human anti-LAG-3antibodies described herein). Additionally or alternatively, theantibody can be a chimeric or humanized antibody.

Similar to its application to tumors as discussed above, antibodymediated LAG-3 blockade can be used alone, or as an adjuvant, incombination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach can be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, & C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. LAG-3 blockade is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of the infections. These novel epitopes are recognized as foreignat the time of anti-human LAG-3 administration, thus provoking a strongT cell response that is not dampened by negative signals through LAG-3.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, LAG-3 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Autoimmune Reactions

Anti-LAG-3 antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489;Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987;Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother EmphasisTumor Immunol 19 (1): 81-4). Therefore, it is possible to consider usinganti-LAG-3 blockade in conjunction with various self proteins in orderto devise vaccination protocols to efficiently generate immune responsesagainst these self proteins for disease treatment. For example,Alzheimer's disease involves inappropriate accumulation of Aβ peptide inamyloid deposits in the brain; antibody responses against amyloid areable to clear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

Other self proteins can also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFα for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-LAG-3 antibody. Neutralizing antibody responses toreproductive hormones can be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors can also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-LAG-3 antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα, and IgE.

Vaccines

Anti-LAG-3 antibodies can be used to stimulate antigen-specific immuneresponses by coadministration of an anti-LAG-3 antibody with an antigenof interest (e.g., a vaccine). Accordingly, in another aspect theinvention provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-LAG-3 antibody, or antigen-binding portionthereof, such that an immune response to the antigen in the subject isenhanced. Preferably, the antibody is a human anti-human LAG-3 antibody(such as any of the human anti-LAG-3 antibodies described herein).Additionally or alternatively, the antibody can be a chimeric orhumanized antibody. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove, or antigens from the viruses, bacteria or other pathogensdescribed above.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of the invention in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-LAG-3 antibodies of the inventioncan be co-administered with one or other more therapeutic agents, e.g.,a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immuno-complex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/ml doseonce every four weeks and adriamycin is intravenously administered as a60-75 mg/ml dose once every 21 days. Co-administration of the humananti-LAG-3 antibodies, or antigen binding fragments thereof, of thepresent invention with chemotherapeutic agents provides two anti-canceragents which operate via different mechanisms which yield a cytotoxiceffect to human tumor cells. Such co-administration can solve problemsdue to development of resistance to drugs or a change in theantigenicity of the tumor cells which would render them unreactive withthe antibody.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain at least oneadditional reagent, or one or more additional human antibodies of theinvention (e.g., a human antibody having a complementary activity whichbinds to an epitope in LAG-3 antigen distinct from the first humanantibody). Kits typically include a label indicating the intended use ofthe contents of the kit. The term label includes any writing, orrecorded material supplied on or with the kit, or which otherwiseaccompanies the kit.

Combination Therapy

In another aspect, the invention provides methods of combination therapyin which an anti-LAG-3 antibody (or antigen-binding portion thereof) ofthe present invention is coadministered with one or more additionalantibodies that are effective in stimulating immune responses to therebyfurther enhance, stimulate or upregulate immune responses in a subject.In one embodiment, the invention provides a method for stimulating animmune response in a subject comprising administering to the subject ananti-LAG-3 antibody and one or more additional immunostimulatoryantibodies, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/oran anti-CTLA-4 antibody, such that an immune response is stimulated inthe subject, for example to inhibit tumor growth or to stimulate ananti-viral response. In another embodiment, the subject is administeredan anti-LAG-3 antibody and an anti-PD-1 antibody. In still anotherembodiment, the subject is administered an anti-LAG-3 antibody and ananti-PD-L1 antibody. In yet another embodiment, the subject isadministered an anti-LAG-3 antibody and an anti-CTLA-4 antibody. In oneembodiment, the anti-LAG-3 antibody is a human antibody, such as anantibody of the disclosure. Alternatively, the anti-LAG-3 antibody canbe, for example, a chimeric or humanized antibody (e.g., prepared from amouse anti-LAG-3 mAb). In another embodiment, the at least oneadditional immunostimulatory antibody (e.g., anti-PD-1, anti-PD-L1and/or anti-CTLA-4 antibody) is a human antibody. Alternatively, the atleast one additional immunostimulatory antibody can be, for example, achimeric or humanized antibody (e.g., prepared from a mouse anti-PD-1,anti-PD-L1 and/or anti-CTLA-4 antibody).

In another embodiment, the invention provides a method for treating ahyperproliferative disease (e.g., cancer), comprising administering aLAG-3 antibody and a CTLA-4 antibody to a subject. In furtherembodiments, the anti-LAG-3 antibody is administered at a subtherapeuticdose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. In anotherembodiment, the present invention provides a method for altering anadverse event associated with treatment of a hyperproliferative diseasewith an immunostimulatory agent, comprising administering an anti-LAG-3antibody and a subtherapeutic dose of anti-CTLA-4 antibody to a subject.In certain embodiments, the subject is human. In other embodiments, theanti-CTLA-4 antibody is human sequence monoclonal antibody 10D1(described in PCT Publication WO 01/14424) and the anti-LAG-3 antibodyis human sequence monoclonal antibody, such as LAG3.5 described herein.Other anti-CTLA-4 antibodies encompassed by the methods of the presentinvention include, for example, those disclosed in: WO 98/42752; WO00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl.Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin.Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr etal. (1998) Cancer Res. 58:5301-5304. In certain embodiments, theanti-CTLA-4 antibody binds to human CTLA-4 with a K_(D) of 5×10⁻⁸ M orless, binds to human CTLA-4 with a K_(D) of 1×10⁻⁸ M or less, binds tohuman CTLA-4 with a K_(D) of 5×10⁻⁹ M or less, or binds to human CTLA-4with a K_(D) of between 1×10⁻⁸M and 1×10⁻¹⁰ M or less.

In another embodiment, the present invention provides a method fortreating a hyperproliferative disease (e.g., cancer), comprisingadministering a LAG-3 antibody and a PD-1 antibody to a subject. Infurther embodiments, the anti-LAG-3 antibody is administered at asubtherapeutic dose, the anti-PD-1 antibody is administered at asubtherapeutic dose, or both are administered at a subtherapeutic dose.In another embodiment, the present invention provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-PD-1 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-1 antibody is a humansequence monoclonal antibody and the anti-LAG-3 antibody is humansequence monoclonal antibody, such as LAG3.5 described herein. Examplesof human sequence anti-PD-1 antibodies include 17D8, 2D3, 4H1, 5C4 and4A11, which are described in PCT Publication WO 06/121168. Otheranti-PD-1 antibodies include, e.g., lambrolizumab (WO2008/156712), andAMP514 (WO2010/027423, WO2010/027827, WO2010/027828, WO2010/098788). Incertain embodiments, the anti-PD-1 antibody binds to human PD-1 with aK_(D) of 5×10⁻⁸ M or less, binds to human PD-1 with a K_(D) of 1×10⁻⁸Mor less, binds to human PD-1 with a K_(D) of 5×10⁻⁹ M or less, or bindsto human PD-1 with a K_(D) of between 1×10⁻⁸M and 1×10⁻¹⁰ M or less.

In another embodiment, the present invention provides a method fortreating a hyperproliferative disease (e.g., cancer), comprisingadministering a LAG-3 antibody and a PD-L1 antibody to a subject. Infurther embodiments, the anti-LAG-3 antibody is administered at asubtherapeutic dose, the anti-PD-L1 antibody is administered at asubtherapeutic dose, or both are administered at a subtherapeutic dose.In another embodiment, the present invention provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-PD-L1 antibody to a subject. In certain embodiments, the subject ishuman. In other embodiments, the anti-PD-L1 antibody is a human sequencemonoclonal antibody and the anti-LAG-3 antibody is human sequencemonoclonal antibody, such as LAG3.5 described herein. Examples of humansequence anti-PD-L1 antibodies include 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT PublicationWO 07/005,874. Other anti-PD-L1 antibodies include, e.g., MPDL3280A(RG7446) (WO2010/077634), MEDI4736 (WO2011/066389), and MDX1105(WO2007/005874). In certain embodiments, the anti-PD-L1 antibody bindsto human PD-L1 with a K_(D) of 5×10⁻⁸M or less, binds to human PD-L1with a K_(D) of 1×10⁻⁸M or less, binds to human PD-L1 with a K_(D) of5×10⁻⁹ M or less, or binds to human PD-L1 with a K_(D) of between1×10⁻⁸M and 1×10⁻¹⁰ M or less.

Blockade of LAG-3 and one or more second target antigens such as CTLA-4and/or PD-1 and/or PD-L1 by antibodies can enhance the immune responseto cancerous cells in the patient. Cancers whose growth may be inhibitedusing the antibodies of the instant disclosure include cancers typicallyresponsive to immunotherapy. Representative examples of cancers fortreatment with the combination therapy of the instant disclosure includethose cancers specifically listed above in the discussion of monotherapywith anti-LAG-3 antibodies.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein can be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-CTLA-4 antibody and an anti-LAG-3 antibody can be administeredsequentially, such as anti-CTLA-4 antibody being administered first andanti-LAG-3 antibody second, or anti-LAG-3 antibody being administeredfirst and anti-CTLA-4 antibody second. Additionally or alternatively, ananti-PD-1 antibody and an anti-LAG-3 antibody can be administeredsequentially, such as anti-PD-1 antibody being administered first andanti-LAG-3 antibody second, or anti-LAG-3 antibody being administeredfirst and anti-PD-1 antibody second. Additionally or alternatively, ananti-PD-L1 antibody and an anti-LAG-3 antibody can be administeredsequentially, such as anti-PD-L1 antibody being administered first andanti-LAG-3 antibody second, or anti-LAG-3 antibody being administeredfirst and anti-PD-L1 antibody second.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof. For example, thefirst administration of a combination anti-CTLA-4 antibody andanti-LAG-3 antibody can be concurrent, the second administration can besequential with anti-CTLA-4 first and anti-LAG-3 second, and the thirdadministration can be sequential with anti-LAG-3 first and anti-CTLA-4second, etc. Additionally or alternatively, the first administration ofa combination anti-PD-1 antibody and anti-LAG-3 antibody can beconcurrent, the second administration can be sequential with anti-PD-1first and anti-LAG-3 second, and the third administration can besequential with anti-LAG-3 first and anti-PD-1 second, etc. Additionallyor alternatively, the first administration of a combination anti-PD-L1antibody and anti-LAG-3 antibody can be concurrent, the secondadministration can be sequential with anti-PD-L1 first and anti-LAG-3second, and the third administration can be sequential with anti-LAG-3first and anti-PD-L1 second, etc. Another representative dosing schemecan involve a first administration that is sequential with anti-LAG-3first and anti-CTLA-4 (and/or anti-PD-1 and/or anti-PD-L1) second, andsubsequent administrations may be concurrent.

Optionally, the combination of anti-LAG-3 and one or more additionalantibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies) can be further combined with an immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines (He et al.(2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccinesthat can be used include peptides of melanoma antigens, such as peptidesof gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cellstransfected to express the cytokine GM-CSF (discussed further below). Acombined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can befurther combined with a vaccination protocol, such as any of thevaccination protocols discussed in detail above with respect tomonotherapy with anti-LAG-3 antibodies.

A combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can alsobe further combined with standard cancer treatments. For example, acombined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can beeffectively combined with chemotherapeutic regimes. In these instances,it is possible to reduce the dose of other chemotherapeutic reagentadministered with the combination of the instant disclosure (Mokyr etal. (1998) Cancer Research 58: 5301-5304). An example of such acombination is a combination of anti-LAG-3 and anti-CTLA-4 antibodiesand/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies further incombination with decarbazine for the treatment of melanoma. Anotherexample is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and/oranti-PD-1 antibodies and/or anti-PD-L1 antibodies further in combinationwith interleukin-2 (IL-2) for the treatment of melanoma. The scientificrationale behind the combined use of LAG-3 and CTLA-4 and/or PD-1 and/orPD-L1 blockade with chemotherapy is that cell death, which is aconsequence of the cytotoxic action of most chemotherapeutic compounds,should result in increased levels of tumor antigen in the antigenpresentation pathway. Other combination therapies that may result insynergy with a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1blockade through cell death include radiation, surgery, or hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors can also be combined with acombined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade. Inhibitionof angiogenesis leads to tumor cell death, which can be a source oftumor antigen fed into host antigen presentation pathways.

A combination of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockingantibodies can also be used in combination with bispecific antibodiesthat target Fcα or Fcγ receptor-expressing effector cells to tumor cells(see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecificantibodies can be used to target two separate antigens. The T cell armof these responses would be augmented by the use of a combined LAG-3 andCTLA-4 and/or PD-1 and/or PD-L1 blockade.

In another example, a combination of anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 antibodies and/or anti-PD-L1 antibodies can be used inconjunction with anti-neoplastic antibodies, such as Rituxan®(rituximab), Herceptin® (trastuzumab), Bexxar® (tositumomab), Zevalin®(ibritumomab), Campath® (alemtuzumab), Lymphocide® (eprtuzumab),Avastin® (bevacizumab), and Tarceva® (erlotinib), and the like. By wayof example and not wishing to be bound by theory, treatment with ananti-cancer antibody or an anti-cancer antibody conjugated to a toxincan lead to cancer cell death (e.g., tumor cells) which would potentiatean immune response mediated by CTLA-4, PD-1, PD-L1 or LAG-3. In anexemplary embodiment, a treatment of a hyperproliferative disease (e.g.,a cancer tumor) can include an anti-cancer antibody in combination withanti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies, concurrently or sequentially or any combination thereof,which can potentiate an anti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992)Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996)Science 274: 1363-1365). In another example, antibodies to each of theseentities can be further combined with an anti-LAG-3 and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 antibody combination to counteractthe effects of immunosuppressive agents and favor anti-tumor immuneresponses by the host.

Other antibodies that can be used to activate host immune responsivenesscan be further used in combination with an anti-LAG-3 and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 antibody combination. These includemolecules on the surface of dendritic cells that activate DC functionand antigen presentation. Anti-CD40 antibodies (Ridge et al., supra) canbe used in conjunction with an anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 combination (Ito et al., supra). Otheractivating antibodies to T cell costimulatory molecules Weinberg et al.,supra, Melero et al. supra, Hutloff et al., supra) may also provide forincreased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being usedto treat a variety of tumors of hematopoietic origin. A combined LAG-3and CTLA-4 and/or PD-1 and/or PD-L1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). These methods can also be used to activateT cell responses to infectious agents such as CMV. Ex vivo activation inthe presence of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 antibodies can be expected to increase the frequency andactivity of the adoptively transferred T cells.

In certain embodiments, the present invention provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease (e.g., cancer) with an immunostimulatoryagent, comprising administering an anti-LAG-3 antibody and asubtherapeutic dose of anti-CTLA-4 and/or anti-PD-land/or anti-PD-L1antibody to a subject. For example, the methods of the present inventionprovide for a method of reducing the incidence of immunostimulatorytherapeutic antibody-induced colitis or diarrhea by administering anon-absorbable steroid to the patient. Because any patient who willreceive an immunostimulatory therapeutic antibody is at risk fordeveloping colitis or diarrhea induced by such an antibody, this entirepatient population is suitable for therapy according to the methods ofthe present invention. Although steroids have been administered to treatinflammatory bowel disease (IBD) and prevent exacerbations of IBD, theyhave not been used to prevent (decrease the incidence of) IBD inpatients who have not been diagnosed with IBD. The significant sideeffects associated with steroids, even non-absorbable steroids, havediscouraged prophylactic use.

In further embodiments, a combination LAG-3 and CTLA-4 and/or PD-1and/or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodiesanti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 antibodies and/or anti-PD-L1antibodies) can be further combined with the use of any non-absorbablesteroid. As used herein, a “non-absorbable steroid” is a glucocorticoidthat exhibits extensive first pass metabolism such that, followingmetabolism in the liver, the bioavailability of the steroid is low,i.e., less than about 20%. In one embodiment of the invention, thenon-absorbable steroid is budesonide. Budesonide is a locally-actingglucocorticosteroid, which is extensively metabolized, primarily by theliver, following oral administration. ENTOCORT EC® (Astra-Zeneca) is apH— and time-dependent oral formulation of budesonide developed tooptimize drug delivery to the ileum and throughout the colon. ENTOCORTEC® is approved in the U.S. for the treatment of mild to moderateCrohn's disease involving the ileum and/or ascending colon. The usualoral dosage of ENTOCORT EC® for the treatment of Crohn's disease is 6 to9 mg/day. ENTOCORT EC® is released in the intestines before beingabsorbed and retained in the gut mucosa. Once it passes through the gutmucosa target tissue, ENTOCORT EC® is extensively metabolized by thecytochrome P450 system in the liver to metabolites with negligibleglucocorticoid activity. Therefore, the bioavailability is low (about10%). The low bioavailability of budesonide results in an improvedtherapeutic ratio compared to other glucocorticoids with less extensivefirst-pass metabolism. Budesonide results in fewer adverse effects,including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004;608-610.

In still further embodiments, a combination LAG-3 and CTLA-4 and/or PD-1and/or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodiesanti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies) in conjunction with a non-absorbable steroid can be furthercombined with a salicylate. Salicylates include 5-ASA agents such as,for example: sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine(DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, SalixPharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & GamblePharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.;ROWASA®, Solvay).

In accordance with the methods of the present invention, a salicylateadministered in combination with anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 antibodies and a non-absorbable steroid canincludes any overlapping or sequential administration of the salicylateand the non-absorbable steroid for the purpose of decreasing theincidence of colitis induced by the immunostimulatory antibodies. Thus,for example, methods for reducing the incidence of colitis induced bythe immunostimulatory antibodies according to the present inventionencompass administering a salicylate and a non-absorbable concurrentlyor sequentially (e.g., a salicylate is administered 6 hours after anon-absorbable steroid), or any combination thereof. Further, accordingto the present invention, a salicylate and a non-absorbable steroid canbe administered by the same route (e.g., both are administered orally)or by different routes (e.g., a salicylate is administered orally and anon-absorbable steroid is administered rectally), which may differ fromthe route(s) used to administer the anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 antibodies.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference. In particular, the disclosures of PCTpublications WO 09/045,957, WO 09/073,533, WO 09/073,546, and WO09/054,863 are expressly incorporated herein by reference.

EXAMPLES Example 1 Design of Variants of LAG3.1 (Antibody 25F7)

Antibody variants of the previously described anti-LAG-3 antibody, 25F7,referred to herein as LAG3.1, were created by first analyzing the aminoacid sequence of the antibody for potential sites of degradation.Expression of site-directed mutagenesis of LAG3.1 V_(H) region wasperformed using QuikChange II XL® Site-Directed Mutagenesis Kit (AgilentTechnologies). The altered V_(H) regions were then subcloned into UCOE®(EMD Millipore) vectors that contain the human IgG4-S228P constantregion. The various heavy chain vectors were each co-transfected with avector expressing the LAG3.1 kappa chain into CHO-S cells, and stablepools were selected for expression.

Five potential deamidation motifs were identified within the variableregion heavy chain CDR2. These sites were located at positions 52, 54,56, 58, and 60 of the heavy chain variable region of LAG3.1 (SEQ ID NO:2) (see FIG. 1A). In particular, deamidation of the “NG” sequence withinthe VH CDR2 (SEQ ID NO: 6) was observed under all conditions, as well asfurther isomerization of the sequence. Deamidation of the startingmaterial was about 10%. Further, it was found that this “NG” sequencedid not correspond to a germline sequence (see FIG. 3). However, theconsensus germline sequence was a potential glycosylation site and,therefore, was not included among the antibody variants.

Four variants (referred to herein as LAG3.5, LAG3.6, LAG3.7, and LAG3.8)were designed which addressed two of the potential deamidation motifs(positions 54 and 56), as shown in FIG. 3. These variants were subjectedto a matrix of conditions as summarized in Table 1 below and thefollowing characteristics were analyzed: (a) chemical and thermalstabilities (physical stability); (b) size exclusion chromatography(aggregation); (c) Isoelectric Focusing gel (IEF) (chargeheterogeneity); (d) activity by Biacore analysis (binding and functionalactivity); and (e) peptide mapping by mass-spectrometry (chemicalmodifications/molecular stability).

TABLE 1 Buffer Acetate (100 nM NaCl, 3% w/v Citrate (100 nM NaCl,mannitol, 0.03% Tween-20) 3% w/v mannitol, 0.03% pH 5.5, 6.0, 6.5, 7.0Tween-20) 5.5, 6.0, 6.5, 7.0 Temperature 4° C. and 37° C. 4° C. and 37°C. Time 0, 4, 8, 12 weeks 0, 4, 8, 12 weeks

Example 2 Characterization of LAG-3 Variants

1. Activated Human CD4⁺ T Cell Binding

To test the ability of the antibody variants to bind to native humanLAG-3 on the surface of activated human T cells, normal healthy donorperipheral blood mononuclear cells were stimulated in 15 cm tissueculture plates at a density of 2×10e6 cells/mL, with a combination ofanti-CD3 (eBioscience, Cat #16-0037-85) and anti-CD28 (BD Bioscience,Cat #555725) antibodies present in solution at 5 μg/mL and 3 μg/mL,respectively. Following three days of stimulation cells were harvested,washed 1× with 1×PFAE buffer (1×PBS+2% FBS, 0.02% sodium azide, 2 mM NaEDTA), and resuspended in 1×PFAE buffer for staining.

For the binding reaction, the LAG3.1 variants were serially diluted withcold 1×PFAE buffer, then 50 μl of diluted antibody solution was mixedwith 50 μl of Fitc-labeled anti-human CD4 (BD Bioscience, Cat #555346)diluted 1:16 in 1×PFAE buffer. For the binding reaction, 100 μl of thisdiluted antibody mixture was added to 2×10⁵ cells and the mixture wasincubated on at 4° C. for 30 minutes. The cells were then washed twotimes with 1×PFAE buffer. A 1:200 dilution of PE-labeled goat anti-humanFcγ-specific antibody (Jackson ImmunoResearch, Cat. #109-116-170) wasadded and the mixture was incubated for 30 minutes at 4° C., followed bywashing twice with cold 1×PFAE buffer. After the final wash, 150 μl ofcold 1×PFAE was added to each solution and analysis of antibody bindingwas carried out by flow cytometry using a FACSCanto flow cytometer (BDBioscience).

The results of the flow cytometry analysis are summarized in FIG. 4Awhich is a graph showing the EC₅₀ for antibody binding to activatedhuman CD4+ T cells. FIG. 4B is a graph showing antibody binding tosoluble human LAG-3/Fc antigen by BIACORE. As shown, the bindingaffinities of LAG3.5 and LAG3.8 are slightly lower, compared to LAG3.1,while their off-rate constants are slightly higher compared to LAG3.1.

2. Physical Stability

Thermal stability and thermal denaturation of the variants was testedusing Microcal VP-DSC. Specifically, each variant was diluted into PBS(Mediatech cat #21-040-CV lot #21040139). The final concentration ofsample was 250 μg/mL after dilution into PBS. The sample was scanned to74° C., cooled to 25° C., and reheated to 74° C. PBS buffer was used asa blank control. Data was fit to a Non-2-state model and curve fittingperformed by Origin software.

As summarized in Table 2 and shown in FIG. 5, LAG3.5 had a highermelting temperature TM2 than LAG3.1, indicating greater overallstability.

TABLE 2 Tm1 (° C.) Tm2 (° C.) Corresponds to CH2 Corresponds to CH3 MAband/or Fab domains and/or Fab domains LAG3.1 70.7 75.7 LAG3.5 70.5 76.3LAG3.6 67.8 70.8 LAG3.7 69.4 73.5 LAG3.8 70.3 75.4

Antibody refolding following denaturation is an inverse measure oflong-term aggregation potential. Accordingly, the LAG-3 variants alsowere tested and compared in terms of thermal reversibility.Specifically, the antibodies were heated to 74° C. and cooled to roomtemperature before heated back to 74° C. The ratio of area under thecurve of the second to first thermograms provides the estimate ofthermal reversibility, which is a direct measure of conformationalreversibility.

As summarized in Table 3 and shown in FIG. 6, LAG3.5 had substantiallyhigher thermal reversibility than all other variants. Notably, thepercent reversibility for LAG3.5 (47%) was more than double that ofLAG3.1 (20%). The thermal reversibility is strongly correlated to thelong-term aggregation potential. Lower reversibility corresponds tohigher potential aggregation. Based on this observation, LAG3.1 wouldpotentially exhibit substantially higher aggregation over time, comparedto LAG3.5. Similarly, all other variants could potentially exhibitsubstantially higher aggregation over time compared to LAG3.5.

TABLE 3 Thermal MAb reversibility (%) LAG3.1 20 LAG3.5 47 LAG3.6 0LAG3.7 11 LAG3.8 26

3. Aggregation

The variants also were tested for stability as a measure of proteinaggregation using standard Size Exclusion HPLC (SEC-HPLC) according thefollowing protocol: antibody test samples were diluted to 1.0 mg/ml withphosphate buffered saline (PBS) and 10 uL was applied to an HPLC(Waters, model 2795). Separation was accomplished on a gel filtrationcolumn (TOSOH Bioscience, TSKgel G3000 SWxl, 7.8 mm×300 mm, product#08541) using a mobile phase of 0.1M sodium phosphate, 0.15M sodiumchloride, 0.1M sodium sulfate, pH 7.2. The analyte was detected bymonitoring UV absorbance at 280 nm, and the antibody peak area percentcomposition was determined using Empower software. As shown in Table 4,LAG3.5 exhibited substantially reduced aggregation compared to LAG3.1.

TABLE 4 IgG Monomer IgG Aggregate Sample (% peak area) (% peak area)LAG3.1 90 10 LAG3.5 96 4 LAG3.6 96 4 LAG3.7 95 5 LAG3.8 95 5

Example 3 Variant Selection

Based on the studies described above, antibody variant LAG3.5 wasselected for further analysis, in view of its significantly improvedphysical and chemical stability compared to its unmodified form(LAG3.1), particularly its high capacity for conformational refolding(thermal reversibility). This analysis included a two-step approach of(a) accelerated stress, (b) followed by 12-week real-time stabilityevaluation. Specifically, LAG3.5 was incubated at 1.0 mg/ml in pH 8.0,50 mM Ammonium Bicarbonate, for 5 days at 40° C. The degree ofmodifications after 5 days was analyzed, as well as the effects onactivity and stability. The LAG3.5 variant was then subjected toreal-time stability in PBS for a duration of 12 weeks and subsequentlyanalyzed. The results of these studies are described below.

1. Antigen Binding

As shown in FIG. 7 (and Table 5), no change in antigen binding wasobserved after 5 days. As also shown in FIGS. 10 A and B, LAG3.5exhibited no change in antigen binding or physical stability after 12weeks. In particular, LAG3.5 maintains higher affinity than LAG3.8 overthe entire 12 week period at both 4° C. and 40° C.

TABLE 5 K_(D) × 10⁻⁹ k_(on) × 10⁴ K_(off) × 10⁻⁴ Clone ID Antigen (M)(1/Ms) (1/s) Lag3.1 PBS 0.21 166 3.44 pH8 0.20 184 3.61 Lag3.5 PBS 0.25130 3.22 pH8 0.20 148 2.98 Lag3.8 PBS 0.25 147 3.68 pH8 0.25 162 4.02

2. Chemical Modifications/Molecular Stability

Peptide mapping by mass spectrometry was used to analyze thechemical/molecular stability of LAG3.5 compared to LAG3.1. Specifically,purified antibody was reduced, alkylated, dialyzed, and digested withtrypsin (Promega Cat. V5111) and GluC (Roche Cat. 11047817001). Digestswere analyzed by nano-LC MSMS mass spectrometry (Thermo Fisher LTQOrbitrap).

As shown in FIG. 8, LAG3.1 showed increased heterogeneity in V_(H)compared to LAG3.5 when subjected to accelerated stability at higher pH,which deamidates asparagine residues (step 1). Change in mass due toisomerization could not be detected under the current experimentalconditions. The percentage change is expressed as a ratio of all changescombined to the parental peak.

In addition, as shown in FIG. 11, LAG3.1 showed increased heterogeneityin V_(H) compared to LAG3.5 when subjected to prolonged real-timestability of 12 weeks, at both 4° C. and 40° C. (step 2).

3. Physical Stability

Thermal reversibility was measured in PBS and at pH 8.0. Under bothconditions, LAG3.5 again exhibited approximately double the level ofrefolding compared to LAG3.1. Specifically, as shown in Tables 6-8,LAG3.5 exhibited 43% refolding compared to 18% for LAG3.1 in PBS. LAG3.5also exhibited 48% refolding compared to 29% refolding for LAG3.1 at pH8.0.

TABLE 6 DSC: melting MAb Condition Tm1 Tm2 Lag3.1 PBS 70.7 75.7 Lag3.1pH8 70.4 75.6 Lag3.5 PBS 70.8 76.4 Lag3.5 pH8 70.5 76.3

TABLE 7 Fluorolog-2: unfolding Mab/mutants Midpoint (M) Aggregation (M)Lag3.1 PBS 1.99 — Lag3.1 pH8 2.08 — Lag3.5 PBS 1.86 — Lag3.5 pH8 2.00 —

TABLE 8 DSC: refolding MAb % reversibility PBS % reversibility pH8Lag3.1 18 29 Lag3.5 43 48

4. Charge Heterogeneity

To assess charge heterogeneity, the variants were analyzed usingisoelectrofocusing (IEF) with standard markers of pI 5.5 and pI 10.0compared to LAG3.1. Briefly, antibody solutions were applied onto a 1 mmthick IEF pI 3-7 pre-made gel (Invitrogen, Cat # EC6648BOX) along with apI 3-10 markers (SERVA, Cat #39212). Electrophoresis was carried outusing IEF 3-7 Cathode buffer (Invitrogen, Cat #LC5370) and IEF Anodebuffer (Invitrogen, Cat # LC5300) and applying electrical current in theorder of 100 V constant for 1 hr, 200 V constant for 1 hr, and 500 Vconstant for 30 min. The IEF gels were stained with Coomassie blue todetect the protein bands and destained with methanol-acetic acidsolution. IEF gels were then analyzed by ImageQuant TL software. Basedon this analysis (data not shown), LAG3.5 exhibited significantly lessheterogeneity compared to LAG3.1.

5. HIC-HPLC

To assess solubility, the variants were analyzed using standardHydrophobic Interaction Chromatography (HIC-HPLC) according to thefollowing protocol: 50 uL of 2M ammonium sulfate was added to 50 uL ofantibody test sample at 1 mg/ml. 80 uL of the test sample was thenapplied to an HPLC (Waters, model 2795) connected in-line to an HICcolumn (TOSOH Bioscience, Ether-5PW TSK-gel, 7.5 mm×75 mm, product#07573). The sample was eluted at a flow rate of 1.0 ml/min with agradient of 100% buffer A (2M ammonium sulfate, 0.1M sodium phosphate,pH 7.0) to 100% buffer B (0.1M sodium phosphate, pH 7.0) over 50minutes. The antibody was detected by monitoring UV absorbance at 280 nmand data was analyzed using Empower software. As shown in FIG. 9, thehydrophilicity of LAG3.5 exhibited solubility at high concentrations ofammonium sulfate.

Example 4 Reversal of T-Cell Mediated Immune Response Inhibition

The activity of LAG3.5 was determined by means of a functional assaythat utilized an antigen-specific mouse T cell hybridoma (3A9).Hybridoma 3A9 expresses a T cell receptor specific for a peptide fromhen egg lysozyme (HEL48-62) and secretes IL-2 when co-cultured withpeptide-pulsed, MHC-matched, antigen presenting cells (LK35.2). SincehuLAG-3-Fc is capable of binding to MHC Class II-positive mouse B celllines, expression of huLAG-3 in the 3A9 line could exert an inhibitoryeffect through engagement with Class II on the murine presenting line. Acomparison of the peptide response profile of the 3A9 parent with thatof the human LAG-3-transduced 3A9 cells co-cultured with MHC-matchedantigen presenting cells demonstrated that the expression of human LAG-3inhibited peptide responsiveness compared to control 3A9 cells. Thisinhibition was reversed by LAG-3 blockade using LAG3.5. Therefore,blockade of LAG-3-mediated inhibition was demonstrated for LAG3.5.

Example 5 T-Cell Activation by LAG3.5

The functional activity of LAG3.5 on primary T cells was assessed usinghuman PBMC cultures stimulated by the superantigen SEB. Total PBMC wereisolated from the blood of eighteen human donors and stimulated for 72hours in either of two assay formats: (i) a fixed amount of antibody (20μg/mL) and serial dilutions of SEB, or (ii) a fixed amount of SEB (85ng/mL) and serial dilutions of antibody. Secreted IL-2, as a measure ofT cell activity, was monitored by ELISA. Antibody anti-PD-1 antibody andIpilimumab were used as positive controls and the activity of LAG3.5 incombination with anti-PD-1 or anti-CTLA-4 was also evaluated for asubset of donors.

Enhanced IL-2 secretion was observed over a range of SEB concentrationsfrom fifteen of the eighteen donors treated with LAG3.5 alone, comparedto isotype control antibody treatment. In most instances the stimulationwas less than that observed for treatment with anti-PD-1 or Ipilimumab.With respect to LAG3.5, the results of the two assay formats (describedabove) were in agreement with one another. Moreover, in 5 of 6 donorstested, combining LAG3.5 with anti-PD-1 or Ipilimumab resulted in higherlevels of stimulation than observed for isotype control antibodycombined with anti-PD-1 or Ipilimumab. These data revealed that LAG3.5can function in normal human T cell assays and can further activateresponses mediated by inhibition of PD-1 and CTLA-4 function.

SUMMARY OF SEQUENCE LISTING SEQ ID NO: DESCRIPTION SEQUENCE  1V_(H) n.a. 25F7 (LAG3.1) >1408_LAG-3_403_25F7.1_VH1_NTCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAATGGAAACACCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCCTATCACTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGTTTGGATATAGTGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  2V_(H) a.a. 25F7 >1408_LAG-3_403_25F7.1_VH1_AAQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHNGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNW FDPWGQGTLVTVSS  3V_(K) n.a. 25F7 >1408_LAG-3_403_25F7.1_VK1_NTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTTGGCCAGGGGACCAACCT GGAGATCAAA  4V_(K) a.a. 25F7 >1408_LAG-3_403_25F7.1_VK1_AAEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK  5V_(H) CDR1 a.a. 25F7 DYYWN  6 V_(H) CDR2 a.a. 25F7 EINHNGNTNSNPSLKS  7V_(H) CDR3 a.a. 25F7 GYSDYEYNWFDP  8 V_(K) CDR1 a.a. 25F7 RASQSISSYLA  9V_(K) CDR2 a.a. 25F7 DASNRAT 10 V_(K) CDR3 a.a. 25F7 QQRSNWPLT 11V_(H) n.a. LAG3.5 V_(H) n.a. LAG3.5caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtgattactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaagcaccaactccaacccgtccctcaagagtcgagtcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggtctgtgaccgccgcggacacggctgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggccagggaaccctggtcaccgtctcctca 12 V_(H) a.a. LAG3.5 V_(H) a.a. LAG3.5QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYS DYEYNWFDPWGQGTLVTVSS13 V_(K) n.a. LAG3.5 V_(K) n.a. LAG3.5gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctctcacttttggccaggggaccaacctggagatcaaa 14V_(K) a.a. LAG3.5 V_(K) a.a. LAG3.5EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQ GTNLEIK 15V_(H) CDR1 a.a. LAG3.5 DYYWN 16 V_(H) CDR2 a.a. LAG3.5 EINHRGSTNSNPSLKS17 V_(H) CDR3 a.a. LAG3.5 GYSDYEYNWFDP 18 V_(K) CDR1 a.a. LAG3.5RASQSISSYLA 19 V_(K) CDR2 a.a. LAG3.5 DASNRAT 20 V_(K) CDR3 a.a. LAG3.5QQRSNWPLT 21 LAG-3 epitope PGHPLAPG 22 LAG-3 epitope HPAAPSSW 23LAG-3 epitope PAAPSSWG 24 V_(H) CDR2 a.a. LAG3.6 EIIHSGSTNSNPSLKS 25V_(H) CDR2 a.a. LAG3.7 EINHGGGTNSNPSLKS 26 V_(H) CDR2 a.a. LAG3.8EINHIGNTNSNPSLKS 27 V_(H) CDR2 a.a. HUMAN GEINHSGSTNY GERMLINE 28(Gly₄-Ser)₃ 29 Human LAG-3 a.a. human LAG-3 a.a. sequenceMWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLTLGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL* 30 V_(H) CDR2 a.a. LAG3.2VIWYDGSNKYYADSVKG 31 V_(H) LAG3.1 n.a. LAG3.1HCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAATGGAAACACCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCCTATCACTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGTTTGGATATAGTGACTACGAGTACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAT GA 32V_(H) LAG3.1 a.a. TRANSLATION\OF\LAG3.1HCQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHNGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* 33 V_(L) LAG3.1 n.a. LAG3.1LCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTTGGCCAGGGGACCAACCTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 34 V_(L) LAG3.1 a.a. TRANSLATION\OF\LAG3.1LCEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* 35 V_(H) LAG3.5 a.a.LAG3.5 heavy chain sequence - completeQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* 36 V_(H) LAG3.5 n.a.LAG3.5 heavy chain sequence - completecaggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtgattactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaagcaccaactccaacccgtccctcaagagtcgagtcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggtctgtgaccgccgcggacacggctgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggccagggaaccaggtcaccgtctcctcagctagcaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga 37 V_(L) LAG3.5 a.a.LAG3.5 kappa chain sequence - CompleteEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC* 38V_(L) LAG3.5 n.a. LAG3.5 - kappa chain sequence - Completegaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctctcacttttggccaggggaccaacctggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

What is claimed:
 1. An isolated monoclonal antibody, or anantigen-binding portion thereof, that binds human LAG-3, comprisingheavy and light chain variable regions, wherein the heavy chain variableregion comprises the CDR1, CDR2, and CDR3 regions from the heavy chainvariable region of SEQ ID NO:
 12. 2. The antibody, or antigen-bindingportion thereof, of claim 1, wherein the heavy chain CDR1, CDR2, andCDR3 regions comprise the amino acid sequences of SEQ ID NOs: 15, 16,and 17, respectively.
 3. The antibody, or antigen-binding portionthereof, of claim 1, wherein the light chain variable region comprisesthe CDR1, CDR2, and CDR3 regions from the light chain variable region ofSEQ ID NO:
 14. 4. The antibody, or antigen-binding portion thereof, ofclaim 3, wherein the light chain CDR1, CDR2, and CDR3 regions comprisethe amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively. 5.The antibody, or antigen-binding portion thereof, of claim 1, whereinthe heavy chain variable region comprises the amino acid sequence of SEQID NO:
 12. 6. The antibody, or antigen-binding portion thereof, of claim1, wherein the light chain variable region comprises the amino acidsequence of SEQ ID NO:
 14. 7. An isolated monoclonal antibody, or anantigen-binding portion thereof, that binds human LAG-3, comprisingheavy and light chain CDR1, CDR2, and CDR3 regions, comprising the aminoacid sequences of SEQ ID NOs: 15, 16, 17, and SEQ ID NOs: 18, 19, and20, respectively.
 8. An isolated monoclonal antibody, or anantigen-binding portion thereof, that binds human LAG-3, comprisingheavy and light chain variable regions, comprising the amino acidsequences of SEQ ID NOs: 12 and 14, respectively.
 9. The antibody, orantigen-binding portion thereof, of claim 1, which exhibits one or acombination of the following properties: (a) binding to monkey LAG-3;(b) lack of binding to mouse LAG-3; (c) binding to LAG-3 majorhistocompatibility (MHC) class II molecules; (d) inhibits binding ofLAG-3 to major histocompatibility (MHC) class II molecules; or (e)stimulates an immune response.
 10. The antibody, or antigen-bindingportion thereof, of claim 1, which stimulates interleukin-2 (IL-2)production in an antigen-specific T cell response.
 11. The antibody, orantigen-binding portion thereof, of claim 1, which stimulates ananti-tumor immune response.
 12. The antibody, or antigen-binding portionthereof, of claim 1, which binds an epitope of human LAG-3 comprisingthe amino acid sequence PGHPLAPG (SEQ ID NO: 21).
 13. The antibody, orantigen-binding portion thereof, of claim 1, which binds an epitope ofhuman LAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 22)or PAAPSSWG (SEQ ID NO: 23).
 14. The antibody, or antigen-bindingportion thereof, of claim 1, which binds to human LAG-3 with a K_(D) of0.27×10⁻⁹ M or less.
 15. The antibody, or antigen-binding portionthereof, of claim 1, which is a human, humanized, or chimeric antibody.16. The antibody, or antigen-binding portion thereof, of claim 1, whichis an IgG1, IgG2 or IgG4 isotype.
 17. The antibody, or antigen-bindingportion thereof, of claim 1, which is an antibody fragment or a singlechain antibody.
 18. A bispecific molecule comprising the antibody, orantigen-binding portion thereof, of claim 1, and a second antibody orantigen-binding portion thereof.
 19. An immunoconjugate comprising theantibody, or antigen-binding portion thereof, of claim 1, linked to atherapeutic agent.
 20. The immunoconjugate of claim 19, wherein thetherapeutic agent is a cytotoxin or a radioactive isotope.
 21. Acomposition comprising the antibody, or antigen-binding portion thereof,of claim 1 and a pharmaceutically acceptable carrier.
 22. A compositioncomprising the bispecific molecule of claim 18 and a pharmaceuticallyacceptable carrier.
 23. A composition comprising the immunoconjugate ofclaim 19, and a pharmaceutically acceptable carrier.
 24. The compositionof claim 21, further comprising an anti-cancer agent.
 25. Thecomposition of claim 24, wherein the agent is an antibody or achemotherapeutic agent.
 26. An isolated nucleic acid encoding the heavyand/or light chain variable region of the antibody, or antigen-bindingportion thereof, of claim
 8. 27. An expression vector comprising thenucleic acid of claim
 26. 28. A host cell comprising the expressionvector of claim
 27. 29. A method for preparing an anti-LAG-3 antibodycomprising expressing the antibody in the host cell of claim 28 andisolating the antibody from the host cell.
 30. A method of stimulatingan immune response in a subject comprising administering the antibody,or antigen-binding portion thereof, of claim 1, to the subject, suchthat an immune response in the subject is stimulated.
 31. The method ofclaim 30, wherein the subject is a tumor-bearing subject and an immuneresponse against the tumor is stimulated.
 32. The method of claim 30,wherein the subject is a virus-bearing subject and an immune responseagainst the virus is stimulated.
 33. The method of claim 30, wherein theimmune response is an antigen-specific T cell response, such that anantigen-specific T cell response is stimulated.
 34. The method of claim33, wherein interleukin-2 production by the antigen-specific T cell isstimulated.
 35. A method for inhibiting growth of tumor cells in asubject comprising administering to the subject the antibody, orantigen-binding portion thereof, of claim 1, such that growth of thetumor is inhibited in the subject.
 36. A method for treating viralinfection in a subject comprising administering to the subject theantibody, or antigen-binding portion thereof, of claim 1, such that theviral infection is treated in the subject.
 37. The method of claim 30,further comprising administration of at least one additionalimmunostimulatory antibody.
 38. The method of claim 37, wherein the atleast one immunostimulatory additional antibody is an anti-PD-1antibody.
 39. The method of claim 38, wherein the at least oneadditional immunostimulatory antibody is an anti-PD-L1 antibody.
 40. Themethod of claim 38, wherein the at least one additionalimmunostimulatory antibody is an anti-CTLA-4 antibody.